The ability of cells to adhere to one another plays a critical role in development, normal physiology, and disease processes such as inflammation. This ability is mediated by adhesion molecules, generally glycoproteins, expressed on cell membranes. Often, an adhesion molecule on one cell type will bind to another adhesion molecule expressed on a different cell type, forming a receptor counter-receptor pair. Three important classes of adhesion molecules are the integrins, selecting, and immunoglobulin (Ig) superfamily members (see Springer, Nature 346:425 (1990); Osborn, Cell 62:3 (1990); Hynes, Cell 69:11 (1992). These molecules are vital to the interaction of leukocytes and platelets with themselves and with the extracellular matrix and vascular endothelium.
The selectin family of receptors are so named because of their lectin-like domain and the selective nature of their adhesive functions. There are three known selecting, L-selectin (also known as LECAM-1, Mel-14 or LAM-1 or CD62L), E-selectin (also called ELAM-1 or CD62E) and P-selectin (also known as CD62, CD62P, GMP140 or PADGEM). The selecting are highly homologous, containing a 120 amino acid (aa) N-terminal lectin domain, an EGF-like domain, a variable number of multiple short consensus repeat (SCR) domains homologous to those found in complement regulatory proteins, followed by a transmembrane domain and short cytoplasmic tail. See Siegelman et al., Science 243:1165-1172 (1989); Lasky et al., Cell 56:1045-1055 (1989); Tedder et al., J. Exp. Med. 170:123-133 (1989); Johnson et al., Cell 56:1033-1044 (1989); Bevilacqua et al., Proc. Natl. Acad. Sci. USA 84:9238-9242 (1987), Bevilacqua et al., Science 243:1160-1165 (1989), Bevilacqua et al., J. Clin. Invest. 91:379-387 (1993), Camerini et al., Nature 280:496-498 (1989). The selecting have overlapping but distinct specificities for counterreceptors. See Bevilacqua et al., J. Clin. Invest. 91:379-387 (1993); Feize, Current Opinion in Struct. Biol. 3:701-710 (1993); Berg et al., Biochem. Biophys. Res. Comm. 184:1048-1055 (1992); Foxall et al., J. Cell Biol. 117:895-902 (1992); Larsen et al., J. Biol. Chem. 267:11104-11110 (1992); Polley et al., Proc. Natl. Acad. Sci. USA 88:6224-6228 (1991) (each of which is incorporated by reference in its entirety for all purposes).
P-selectin is constitutively expressed by both platelets and endothelial cells where it is stored in .alpha.-granules or Weibel-Palade bodies for rapid (seconds to minutes) translocation to the cell surface upon activation by, for example, thrombin or histamine (McEver et al., J. Biol. Chem. 250:9799-9804 (1984); Hsu-Lin et al., J. Biol. Chem. 264:8121-9126 (1984)). E-selectin is expressed by activated endothelial cells (e.g., after TNF-.alpha. or IL-1 stimulation for 6-8 hr). Its expression is controlled at the transcriptional level (Bevilacqua et al., 1987, supra; Bevilacqua et al., 1989, supra). P-selectin and E-selectin both bind to neutrophils and monocytes (Larsen et al., Cell 59:305-312 (1989); Johnston et al., Cell 56:1033-1044 (1989); Bevilacqua et al., 1987, supra; Bevilacqua et al., 1989, supra), as well as subsets of lymphocytes (Picker et al., Nature 349:796-799 (1991); Shimizu et al., Nature 349:799-802 (1991); Moore et al., BBRC 186:173-181 (1992)). L-selectin is constitutively expressed by leukocytes, and mediates lymphocyte adhesion to peripheral lymph node high endothelial venules (HEV) (Gallatin et al., Nature 304:30-34 (1983); Berg et al., Immunol. Rev. 108:5-18 (1989); Berg et al., J. Cell. Biol. 114:343-349 (1991)), and neutrophil adhesion to cytokine-activated endothelial cells (Hallman et al., Biochem. Biophys. Res. Comm. 174:236-243 (1991); Smith et al., J. Clin. Invest. 87:609-618 (1991); Spertini et al., J. Immunol. 147:2565-2573 (1991)). L-selectin is a counter-receptor on neutrophils for both E-selectin and P-selectin (Kishimoto et al., Blood 78:805-811 (1990), Picker et al., Cell 66:921 (1991)), although all three selectins probably have other counter-receptors as well.
E-selectin, P-selectin and L-selectin mediate leukocyte-endothelial cell and platelet-leukocyte adhesive interactions during inflammation (Bevilacqua et al., 1993, supra). All three selectins have been demonstrated to participate in an initial "rolling" interaction of leukocytes with activated endothelium (von Andrian et al., Proc. Natl. Acad. Sci. USA 88:7538-7542 (1991); Ley et al., Blood 77:2553-2555 (1991); Abassi et al., J. Clin. Invest. 92:2719-2730 (1993); Dore et al., Blood 82:1308-1316 (1993); Jones et al., Biophys. J. 65:1560-1569 (1993); Mayadas et al., Cell 74:541-554 (1993)). This initial interaction precedes CD18-integrin-mediated adhesion and subsequent migration of neutrophils through the endothelium and into inflamed tissue sites (Lawrence et al., Cell 65:859-873 (1991); von Andrian et al., Am. J. Physiol. 263:H1034-H1044 (1992)). Depending on the nature of inflammatory stimuli and time after initiation of inflammatory response, either E-selectin or P-selectin may be functionally dominant in promoting neutrophil-mediated tissue damage.
In principle, antibodies or other antagonists of the selecting could abort the adhesion process, thereby preventing neutrophils from binding to the endothelium and from extravasating into tissues. A substantial number of antibodies specific for one of the selecting have been reported. Some of these antibodies have been reported to block binding of selecting to counterreceptors in vitro. Some of the antibodies have also been reported to block selectin-mediated interactions in animal models in vivo. For example, antibodies to E-selectin have been reported to protect against neutrophil-mediated damage in an IgG complex model of lung injury in the rat (Mulligan et al., J. Clin. Invest. 88:1396 (1991)). Antibodies to P-selectin have been reported to protect against acute lung injury induced by intravenous injection of cobra venom factor (Mulligan et al., J. Clin. Invest. 90:1600-1607 (1992)), as well as in a rat model of systemic endotoxemia (Coughlan et al., J. Exp. Med. 179:329-334 (1994)). Antibodies to P-selectin have also been reported to be protective in a cat model of myocardial ischemia and reperfusion injury (Weyrich et al., FASEB J. 7:A785 (1993)).
Although some antibodies against E-selectin and P-selectin have shown blocking activity, many, if not most, antibodies specific for E-selectin or P-selectin are nonblocking (see, e.g., Bevilacqua et al., 1989, supra; Erbe et al., J. Cell Biol. 119:215-227 (1992)). That is, these antibodies bind to epitopes in the extracellular domains of E-selectin or P-selectin that do not directly participate in counterreceptor binding or the subsequent cellular adhesion process. The prevalence of nonblocking antibodies suggests that only small regions of the extracellular domain participate directly in binding or influence binding. Thus, de novo screening of antibodies generated against E-selectin or P-selectin would be expected to generate mainly nonblocking antibodies.
Despite the large number of antibodies isolated to-date against the three selectins, there have been few reports of crossreacting antibodies that bind to more than one selectin. Crossreacting antibodies might be capable of aborting the inflammatory process at more than one level, thereby providing more broadly useful therapeutic agents for neutrophil-mediated inflammatory conditions than antibodies specific for a single selectin. One antibody has been reported to crossreact with human E-selectin and dog L-selectin but not with the two selectins from the same species (Abassi et al., J. Immunol. 147:2107-2115 (1991)). A second antibody has been reported to crossreact with human E-selectin and L-selectins (Jutila et al., J. Exp. Med. 175:1565-1573 (1992); WO/9324614). However, no antibody has been isolated that binds to both P-selectin and E-selectin, much less blocks the functions of both of these molecules.
Accordingly, there is a need for antibodies that bind to both E-selectin and P-selectin, preferably so as to block the capacity of both of these molecules to participate in adhesion reactions with counterreceptors. The present invention fulfills this and other needs.